The resurgence of mosquito-borne diseases places a growing importance on research that will better delineate mechanisms influencing vector competence. Following ingestion with a blood meal, all mosquito-borne pathogens must survive the midgut environment and traverse the peritrophic matrix (PM) and midgut epithelium to reach specific tissues required for development and/or transmission; therefore the PM serves as a barrier that must be circumvented if a pathogen is to be successfully transmitted. The PM is a proteoglycan matrix composed of chitin, proteins and glycoproteins that is produced by the midgut and serves to separate the blood bolus from the midgut epithelium. The focus of this research proposal is aimed at increasing our understanding of midgut chitin biosynthesis required for PM formation in the mosquito, Aedes aegypti. During the previous period of support, studies were initiated on three key enzymes to verify their involvement in chitin synthesis and to begin an assessment of factors controlling their expression. It now is known that glutamine synthetase (GS), glucosamine: fructose-6-phosphate amidotransferase (GFAT) and chitin synthase (CS) play major roles in chitin synthesis in the midgut. A major hurdle to detailed studies of factors responsible for the blood meal-induced upregulation of expression of these genes in midguts has been the protease-induced degradation of nuclear proteins during extraction. During the initial period of support, experimental protocols were developed to alleviate this problem; consequently, electrophoretic mobility shift assays (EMSA) now have identified several important cis-acting elements and putative trans-acting factors that bind those elements and selectively control GS expression in midgut tissues. However, It still is not known how chitin is trafficked from the cytoplasm of midgut cells to the extracellular space of the midgut lumen where PM formation occurs. Likewise, three additional enzymes, glucosamine-6-phosphate N-acetyltransferase (GNAT), phosphoacetyl glucosamine mutase (AGM), and uridine diphosphate-N-acetylglucosamine pyrophophrylase (UAP) are also involved in this pathway but have never been cloned or characterized in any insect. In this competing continuation application various microscopical, biochemical and molecular techniques will be used with the mosquito, Ae. aegypti, to extend studies on the chitin biosynthetic pathway by (1) cloning and characterizing the genes encoding GNAT, AGM, and UAP and determining temporal and spatial transcription and translation in midguts following blood feeding, (2) identifying key regulatory elements in the GS, GFAT and CS genes that are responsible for midgut-specific up regulation of expression following a blood meal, (3) performing gene silencing studies, using a transducing virus system, to obtain direct evidence of the roles these enzymes play in chitin biosynthesis, and (4) obtaining structure-function information to evaluate how chitin is trafficked out of midgut cells and also mechanistically determining the regulatory function of GFAT in feedback inhibition of chitin synthesis.